Caspases play important roles in multiple cellular processes, including apoptosis, cell growth and proliferation, and cytokine maturation. Caspases at the top of the apoptosis signaling cascade are called initiator caspases. They are critical in inducing the onset of apoptosis signaling. Currently, the precise molecular mechanisms for the activation of initiator caspases have not been resolved. Moreover, it has been difficult to assess the in vivo functions of these initiator caspases because their knockouts in mice cause embryonic lethality. In this proposal, experiments are proposed to study the molecular mechanisms of activation and in vivo functions of initiator caspases: 1) To test the mechanism of dimer-dependent caspase activation. Crystal structural studies suggest that caspases form homodimers prior to their autoactivation. We will use an inducible dimeric system to study how the two caspase molecules in the dimer interact to cause caspase activation. 2) To examine the caspase activation cascades. Caspases may form a signaling cascade to transmit apoptosis signals. The interactions between different caspases in the signaling cascade during apoptosis are not well understood. We will study whether two different caspases interact at the procaspase level and in apoptosis signaling cascade. 3) To dissect the functions of caspase-8 and caspase-9 in conditional knockout models. Caspase-8 and caspae-9 are the initiator caspases in death receptor- and mitochondrion-dependent apoptosis, respectively. However, conventional knockouts of caspase-8 and caspase-9 result in embryonic or perinatal lethality, making it difficult to assess the in vivo functions of these caspases. We will generate conditional knockouts of caspase-8 and caspase-9 in mice in order to study their functions in vivo: These studies will help to elucidate the mechanisms for caspase activation and the functions of pro-apoptotic caspases in regulating T cell functions in vivo, thereby facilitating the design of therapeutic approaches to target these caspases in autoimmune diseases and cancer.